Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Psoralen is a linear three ring heterocyclic compound having the structure ##STR2##
It is a bifunctional photoreactive molecule which forms covalent bonds with nucleic acids in the presence of near ultraviolet (UV) light (1). For a review of psoralen photochemistry, see Hearst (2).
Psoralen's ability to react with DNA has given it clinical importance in the treatment of psoriasis and other skin disorders. Additionally, its ability to form interstrand crosslinks in double stranded DNA has made it a useful reagent in the study of nucleic acid structure and function. Biotin, a growth factor present in very minute amounts in every living cell and occurring mainly bound to proteins or polypeptides, has the structure ##STR3## Avidin is a glycoprotein containing four essentially identical subunits, each of which is a single polypeptide chain of 128 amino acid residues with a carbohydrate moiety attached at position 17 (3).
Biotin combines with avidin and becomes inactive (4, 5). The exceptionally high affinity of avidin for biotin has provided the basis for the development of sensitive and specific detection systems. Typically, in a multi-step procedure, antigens are recognized by specific antibodies, which are then bound to a biotinylated anti-immunogloulin. This then forms a tight complex with avidin, either conjugated directly to a signal of some kind, such as a fluorescent dye or enzyme, or in turn bound to a biotinylated label. Alternatively, molecules that are themselves directly biotinylated can be recognized directly by avidin, omitting the antibody reactions, with their somewhat lower affinities.
In 1985, G. D. Cimino et al. (6) described the synthesis of a psoralen derivative (aminomethyltrioxsalen-AMT) which contains a biotin moiety attached to the 4' position by various undisclosed linker chains. However, the purported methods for synthesizing these compounds were not disclosed. Furthermore, Cimino et al. reported only that preliminary studies of these undisclosed compunds indicated that they could be used to interchalate and crosslink double-stranded nucleic acid and that they can be detected colorimetrically or fluorescently by standard methods based on the avidin-biotin interaction.
Presently, biotinylated psoralens which retain the biological activity of psoralen and the binding specificity of biotin for avidin are not known. Furthermore, methods for synthesizing compounds which retain the biological activity of psoralen and the binding specificity of biotin are not known. A quick, easy, efficient, and safe method for preparing a biotinylated psoralen would provide readily accessible amounts of biotinylated psoralens useful for psoralen modification of cellular components, the visualization of minute amounts of DNA, investigations of the uptake and distribution of psoralen within cells, the delivery of psoralen to specific cells, and the conversion of nucleic acid molecules to ligands for avidin.